scholarly journals Activation of platelets by alpha-thrombin is a receptor-mediated event. D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone-thrombin, but not N alpha-tosyl-L-lysine chloromethyl ketone-thrombin, binds to the high affinity thrombin receptor.

1986 ◽  
Vol 261 (34) ◽  
pp. 15928-15933 ◽  
Author(s):  
J T Harmon ◽  
G A Jamieson
Keyword(s):  
Thrombin Receptor ◽  
Chloromethyl Ketone ◽  
High Affinity

Role of external Na+ and cytosolic pH in agonist-evoked cytosolic Ca2+ response in human platelets

1994 ◽  
Vol 267 (6) ◽  
pp. C1543-C1552 ◽  
Author(s):  
M. Kimura ◽  
K. Nakamura ◽  
J. W. Fenton ◽  
T. T. Andersen ◽  
J. P. Reeves ◽  
...  
Keyword(s):  
Binding Sites ◽  
Human Platelets ◽  
Thrombin Receptor ◽  
Free Medium ◽  
Amidolytic Activity ◽  
Cytosolic Ph ◽  
Amino Acid Peptide ◽  
High Affinity ◽  
Inositol 1,4,5 Trisphosphate

The role of external Na+ in agonist-evoked platelet Ca2+ response is poorly understood. This was explored in this study. Removal of external Na+ decreased both cytosolic Ca2+ mobilization and external Ca2+ entry, induced by thrombin but not by ADP or vasopressin. That external Na+ regulates thrombin activities was demonstrated by 1) Na+ dependency of the amidolytic activity of thrombin, 2) inhibition of thrombin binding to the high-affinity binding sites in Na(+)-free medium, and 3) attenuation of thrombin-induced inositol 1,4,5-trisphosphate production in Na(+)-free medium. Moreover, Ca2+ response to the thrombin receptor 6-amino acid peptide was independent of external Na+. The role of external Na+ in modifying agonist-evoked Ca2+ response through activation of Na+/H+ antiport and cytosolic alkalinization was then explored. Cytosolic alkalinization by monensin or NH4Cl enhanced thrombin, ADP, and thimerosal-induced external Ca2+ entry. Thimerosal-induced acceleration of external Ca2+ entry was diminished by the inhibition of Na+/H+ antiport. Thus external Na+ enhances thrombin activities, and cytosolic pH mediates store-regulated external Ca2+ entry. However, Na+/H+ antiport activation is not essential for agonist-evoked Ca2+ mobilization and external Ca2+ entry.

scholarly journals Monoclonal antibody to the thrombin receptor stimulates DNA synthesis in combination with gamma-thrombin or phorbol myristate acetate.

1987 ◽  
Vol 105 (6) ◽  
pp. 2551-2558 ◽  
Author(s):  
G H Frost ◽  
W C Thompson ◽  
D H Carney
Keyword(s):  
Monoclonal Antibody ◽  
Cell Proliferation ◽  
Dna Synthesis ◽  
Human Fibroblasts ◽  
Enzymic Activity ◽  
Thrombin Receptor ◽  
High Affinity ◽  
Thrombin Receptors ◽  
Affinity Interaction

Studies with various thrombin derivatives have shown that initiation of cell proliferation by thrombin requires two separate types of signals: one, generated by high affinity interaction of thrombin or DIP-thrombin (alpha-thrombin inactivated at ser 205 of the B chain by diisopropylphosphofluoridate) with receptors and the other, by thrombin's enzymic activity. To further study the role of high affinity thrombin receptors in initiation, we immunized mice with whole human fibroblasts and selected antibodies that blocked the binding of 125I-thrombin to high affinity receptors on hamster fibroblasts. One of these antibodies, TR-9, inhibits from 80 to 100% of 125I-thrombin binding, exhibits an immunofluorescent pattern indistinguishable from that of thrombin bound to receptors on these cells, and selectively binds solubilized thrombin receptors. By itself, TR-9 did not initiate DNA synthesis nor did it block thrombin initiation, but TR-9 addition to cells in the presence of alpha-thrombin, gamma-thrombin (0.5 microgram/ml), or PMA stimulated thymidine incorporation up to threefold over controls. In all cases, maximal stimulation was observed at concentrations of TR-9, ranging from 1 to 4 nM corresponding to concentrations required to inhibit from 30 to 100% of 125I-thrombin binding. These results demonstrate that the binding of the monoclonal antibody to the alpha-thrombin receptor can mimic the effects of thrombin's high affinity interaction with this receptor in stimulating cell proliferation.

Platelet activation by thrombin in the absence of the high-affinity thrombin receptor

Biochemistry ◽  
1988 ◽  
Vol 27 (6) ◽  
pp. 2151-2157 ◽  
Author(s):  
Joan T. Harmon ◽  
G. A. Jamieson
Keyword(s):  
Platelet Activation ◽  
Thrombin Receptor ◽  
High Affinity

High-affinity thrombin receptor (PAR-1) ligands: a new generation of indole-based peptide mimetic antagonists with a basic amine at the C-terminus

2003 ◽  
Vol 13 (13) ◽  
pp. 2199-2203 ◽  
Author(s):  
Han-Cheng Zhang ◽  
Kimberly B. White ◽  
David F. McComsey ◽  
Michael F. Addo ◽  
Patricia Andrade-Gordon ◽  
...  
Keyword(s):  
Thrombin Receptor ◽  
High Affinity ◽  
Peptide Mimetic ◽  
C Terminus ◽  
New Generation ◽  
Basic Amine

Receptor mechanism of thrombin-mediated pulmonary vasodilation in neonates

1993 ◽  
Vol 265 (4) ◽  
pp. L355-L359
Author(s):  
J. M. Pinheiro ◽  
T. T. Andersen ◽  
A. B. Malik
Keyword(s):  
Peptide Sequence ◽  
Thrombin Receptor ◽  
Albumin Solution ◽  
Vascular Effects ◽  
Chloromethyl Ketone ◽  
Cellular Effects ◽  
Pulmonary Vasodilation ◽  
Pulmonary Vasodilator ◽  
Potent Vasodilator ◽  
Proteolytic Action

A recently identified peptide sequence exposed after proteolytic cleavage of the NH2-terminus of the thrombin receptor mimics some cellular effects of alpha-thrombin. To determine whether a proteolytic action of thrombin is required for vasoactivity, we examined the vascular effects of modified thrombins and synthetic NH2-terminus peptide sequences of the thrombin receptor (TRPs) in isolated piglet lungs. Lungs of piglets 1-6 days old were perfused with recirculating Ringer-albumin solution at a constant flow of 60 ml/min. We measured the pulmonary artery pressure (Ppa) and segmental distribution of pulmonary vascular resistance (using the double occlusion method) in response to injections of human alpha-thrombin, modified thrombins, and TRP-14 and TRP-7 (i.e., 14 and 7 amino acid NH2-terminus peptides of the cleaved thrombin receptor). alpha-Thrombin produced a rapid and transient decrease in Ppa; the magnitude and duration [time for one-half recovery (t1/2 R)] of the vasodilation responses were concentration dependent [t1/2 R values of 1.4 +/- 0.1 and 3.3 +/- 2.4 min (mean +/- SE) at concentrations of 10(-10) and 10(-9) M, respectively]. The vasodilation was due primarily to a decrease in precapillary resistance. Proteolytically active, but binding-impaired gamma-thrombin was a less potent vasodilator and proteolytically inactive D-phenylalanyl-prolyl-arginine-chloromethyl ketone (PPACK)-alpha-thrombin did not induce vasodilation. TRP-14 was also a pulmonary vasodilator with a t1/2R value of 0.8 +/- 0.09 min at a concentration of 10(-7) M; both TRP-14 and TRP-7 were approximately 3-log less potent than equimolar alpha-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

PATHOGENIC BACTERIA HAVE HIGH AFFINITY RECEPTORS SPECIFIC FOR PLASMIN

1987 ◽  
Author(s):  
T A Broeseker ◽  
M D P Boyle ◽  
R Lottenberg
Keyword(s):  
Pathogenic Bacteria ◽  
Aminocaproic Acid ◽  
Fibrin Clot ◽  
Chloromethyl Ketone ◽  
High Affinity ◽  
Data Binding ◽  
Binding Data ◽  
Group A ◽  
Human Plasminogen ◽  
Epsilon Aminocaproic Acid

Binding of the key fibrinolytic enzyme, plasmin, to certain pathogenic group A streptococci was studied. In these experiments the ability of a group A streptococcal strain, 64/14, to bind either 125I-human plasminogen or the same label following activation with urokinase was measured. It was found that this strain bound <10% of the labeled plasminogen but >70% of labeled plasmin. This property distinguishes the plasmin receptor from streptokinase. These bacteria did not express a common serine protease receptor/inhibitor since they failed to bind labeled trypsin or urokinase. Maximal binding of plasmin occurred between pH 6.0 and 8.0 and in the ionic strength range of 50-200 mM salt. The Kd of plasmin binding to bacteria was approximately 10-10 M at pH 7.4 in 150 mM salt. This was determined by a non-linear least squares analysis of equilibrium binding data. Binding was reversibly inhibited by either epsilon aminocaproic acid (I50 of 0.2 mM) or lysine (I50 of 3.0 mM) suggesting the involvement of the high affinity lysine binding site of plasmin in its binding to bacteria. Bacterial bound plasmin retains its enzymatic activity, being capable of cleaving chromogenic substrates and solubilizing a fibrin- clot. The bacterial bound enzyme activity was inhibited by the low molecular weight inhibitors aprotinin and phe-pro-arg chloromethyl ketone but not by alpha-2 plasmin inhibitor. The ability of bacteria to acquire membrane associated proteolytic activity which cannot be physiologically inhibited may contribute to their tissue invasive properties.

scholarly journals Role of Glycoprotein V in the Formation of the Platelet High-Affinity Thrombin-Binding Site

Blood ◽  
1997 ◽  
Vol 89 (12) ◽  
pp. 4355-4363 ◽  
Author(s):  
Jing-Fei Dong ◽  
Garunee Sae-Tung ◽  
José A. López
Keyword(s):  
Binding Site ◽  
Surface Expression ◽  
Thrombin Receptor ◽  
Platelet Surface ◽  
High Affinity ◽  
L Cells ◽  
High Affinity Binding Site ◽  
Physiologic Function ◽  
Von Willebrand

Abstract The glycoprotein (GP) Ib-IX-V complex contains a high-affinity binding site for thrombin on the platelet surface with a poorly defined role in platelet activation by this agonist. Four polypeptides comprise the complex: GP Ibα, GP Ibβ, GP IX, and GP V. The site within the complex that binds thrombin has been localized to a 45-kD region at the amino terminus of GP Ibα, which also contains the site through which the complex interacts with von Willebrand factor. A GP Ib-IX complex that lacks GP V can be efficiently expressed on the surface of transfected cells. We examined the ability of L cells expressing the GP Ib-IX complex (L2H cells) to bind thrombin at high affinity, and found no increase over the level of thrombin binding to control L cells. Because it is one of the few substrates for thrombin on the platelet surface, GP V has also been implicated as possibly participating in thrombin's actions on the platelet. To examine the role of GP V in forming the high-affinity thrombin-binding site, we compared the binding of thrombin to L2H cells versus cells that express the entire GP Ib-IX-V complex (L2H/V cells). Surface expression of GP Ibα was equivalent in these two stable cell lines. Thrombin binding to L2H/V cells was detectable at 0.25 nmol/L thrombin and reached a plateau at 1 nmol/L. No binding to L2H cells was detectable at these concentrations. Comparable results were obtained when thrombin binding to L2H cells transiently expressing GP V was compared with its binding to sham-transfected L2H cells. Again, only cells transiently expressing GP V bound thrombin specifically. As with the platelet polypeptide, thrombin cleaved GP V from the surface of L2H/V cells. To test whether GP V cleavage was required for enhancing thrombin binding to the complex, we tested the binding of enzymatically inactive D-phenylalanyl-Lprolyl-L-arginine chloromethylketone (PPACK)-thrombin to L2H and L2H/V cells. Like native thrombin, PPACK-thrombin at 1 nmol/L bound only to L2H/V cells, indicating that GP V cleavage is not a prerequisite for the formation of the high-affinity thrombin receptor. These data provide the first indication of a physiologic function for GP V, and suggest that formation of the high-affinity thrombin receptor on the platelet surface has complex allosteric requirements.

The Anion-Binding Exosite is Critical for the High Affinity Binding of Thrombin to the Human Thrombin Receptor

1994 ◽  
Vol 11 (1) ◽  
pp. 17-28 ◽  
Author(s):  
Brian D. Blackhart ◽  
Grace Cuenco ◽  
Timothy Toda ◽  
Robert M. Scarborough ◽  
David L. Wolf ◽  
...  
Keyword(s):  
Anion Binding ◽  
Thrombin Receptor ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Human Thrombin

Thrombomodulin: An overview and potential implications in vascular disorders

Lupus ◽  
1998 ◽  
Vol 7 (2_suppl) ◽  
pp. 120-125 ◽  
Author(s):  
M-C Boffa ◽  
M Karmochkine
Keyword(s):  
Protein C ◽  
Inflammatory Diseases ◽  
Predictive Marker ◽  
Endothelial Damage ◽  
Diabetic Microangiopathy ◽  
Enzyme Linked Immunosorbent Assay ◽  
Thrombin Receptor ◽  
High Affinity ◽  
Rickettsial Infections ◽  
Activated Neutrophils

Thrombomodulin (TM), a high affinity thrombin receptor present on endothelial cell membrane, plays an important role as a natural anticoagulant. It acts as a cofactor of thrombin-catalyzed activation of protein C, and inhibits the procoagulant functions of thrombin. TM is also located in other cells (keratinocytes, osteoblasts, macrophages, …) where it might be involved in cell differentiation or in inflammation. In the presence of cytokines, activated neutrophils and macrophages, endothelial TM is cleaved enzymatically, releasing soluble fragments which circulate in the blood and are eliminated in urine. Plasma TM level (pTM) can be measured using a two-site enzyme-linked immunosorbent assay (ELISA). pTM level is regarded as a molecular marker reflecting injury of endothelial cells. It is often increased in case of diffuse endothelial damage as in disseminated intravascular coagulation, diabetic microangiopathy, Plasmodium falciparum and rickettsial infections. pTM is also a predictive marker of hypertensive complications in pregnancy. In several systemic inflammatory diseases, pTM levels are correlated to the activity of the disease.

Both the High Affinity Thrombin Receptor (GPIb-IX-V) and GPIIb/IIIa Are Implicated in Expression of Thrombin-induced Platelet Procoagulant Activity

2001 ◽  
Vol 86 (10) ◽  
pp. 1065-1069 ◽  
Author(s):  
Donna Pedicord ◽  
Dietmar Seiffert ◽  
G.A. Jamieson ◽  
Nicholas Greco ◽  
Ira Dicker
Keyword(s):  
Platelet Aggregation ◽  
Procoagulant Activity ◽  
Thrombin Receptor ◽  
High Affinity ◽  
Normal Platelet

SummaryPlatelets activated by -thrombin express surface procoagulant activity (PCA) that accelerates the conversion of prothrombin to α-thrombin. Following activation with 10 nM α-thrombin, the PCA of normal platelets was approximately five-fold higher than that of Bernard-Soulier platelets (lacking GPIb). Normal platelet PCA was inhibited ~50 % by activation in the presence of the anti-GPIb MoAbs LJIb10 or TM60. Moreover, normal platelet PCA was completely abrogated in the presence of a combination of both LJIb10 and c7E3, a MoAb directed against αIIbβ3 (GPIIb/IIIa). In contrast, PCA expressed by Bernard Soulier or Glanzmann platelets was not inhibited by either LJIb10 or c7E3 MoAb. The platelet activating peptide SFLLRN at 10 μM, a concentration which fully activates platelet aggregation and Ca2+ mobilization, generated PCA activity one fifth of that generated by α-thrombin at 10 nM but anti-PAR1 antibodies did not affect thrombin-induced PCA expression. These results demonstrate that GPIb mediates, at least in part, the thrombin-induced activation of platelets that leads to PCA, and that αIIbβ3 is also involved in PCA generation, but these results do not support a major role for PAR1 in this activation.

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